The surface markers of cells were determined by direct immunofluorescence staining and analysed by flowcytometer. All antibodies were listed in Table 1. Cells were harvested and washed with 0.5% of BSA in 1X PBS (phosphate-buffered saline). Aliquots of cells from 105 to 106 were labelled with anti-human monoclonal antibodies, for 20 minutes at 2-8oC and washed with 0.5% of BSA in 1X PBS. All antibodies are raised in mice against human epitopes. The fluorochrome analysis wa...

... middle of paper ...

...idine (3H-TdR) incorporation assays

Proliferation assays were measured by tritium thymidine (3H-TdR) incorporation which reveals the proportion of cells in S phase of the cell cycle. In brief, at final 18 hours of incubation, 3H-TdR (0.037 MBq/well [0.5 μCi/well]) (Pelkin Elmer) were added to pulse the cultures. At the end of incubation, the cells were harvested onto glass fibre filter mats A (Perkin Elmer) using a 96 well plate manual cell harvester (MACH IIIM-FM, Tomtec, Inc. Hamden, CT USA). Scintillation cocktail was added to amplify the signal. Liquid scintillation spectroscopy was counted by Microbeta Trilux beta counter (Pelkin Elmer).

3.6 Statistical Analysis

Values for all measurements are presented as mean ± SD or stated otherwise. Comparisons for all pairs were performed by Student’s t-test. Significance levels were set at p value of 0.05.

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